LOCUS       sequence-490441-        7787 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  This plasmid will be used to replace the promoter of a gene with the
            PenotetSW2 promoter at its endogenous locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7787)
  AUTHORS   Bashir S, Sivakumar S, Rhind N
  TITLE     A Tetracycline-Inducible Promoter Replacement System for 
            Schizosaccharomyces pombe.
  JOURNAL   MicroPubl Biol. 2025 Aug 21;2025:10.17912/micropub.biology.001759. 
            doi: 10.17912/micropub.biology.001759. eCollection 2025.
  PUBMED    40918427
REFERENCE   2  (bases 1 to 7787)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MicroPubl 
            Biol."; date: "2025-08-21"; volume: 
            "2025:10.17912/micropub.biology.001759. doi"; pages: " 
            10.17912/micropub.biology.001759. eCollection 2025"
COMMENT     Sequence Label: pFS563
FEATURES             Location/Qualifiers
     source          1..7787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1135..1155)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1750..1778
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    1871..1889
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        2197..2576
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2577..2780
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2731..2751
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2777..2801
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2882..3505
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2955..2974)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3515..3533)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3543..3559)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3728..4847)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3728..3925)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3931..4503)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4504..4847)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(4991..5009)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5016..5032)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5173..5601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5260..5279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5470..5491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             5945..6739
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5999..6018)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6609..6628
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6757..6825,6826..7617)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6975..6994)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg gtaccgagct
     1021 cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttgaattc gagctcgttt
     1081 aaactgtaag atcttttgta tagttcatcc atgccatgtg taatcccagc agctgttaca
     1141 aactcaagaa ggaccatgtg gtctctcttt tcgttgggat ctttcgaaag cttagattga
     1201 taggacaggt aatggttgtc tggtaaaagg acagggccat cgccaattgg agtattttgt
     1261 tgataatggt ctgctagttg aacgcttcca tcttcaatgt tgtgtctaat tttgaagtta
     1321 actttgattc cattcttttg tttgtctgcc atgatgtata cattgtgtga gttatagttg
     1381 tattccaatt tgtgtccaag aatgtttcca tcttctttaa aatcaatacc ttttaactcg
     1441 attctattaa caagggtatc accttcaaac ttgacttcag cacgtgtctt gtagttcccg
     1501 tcatctttga aaaatatagt tctttcctgt acataacctt cgggcatggc actcttgaaa
     1561 aagtcatgcc gtttcatatg atctgggtat ctagcaaagc acattagacc ataacccaaa
     1621 gtagtgacaa gtgttggcca tggaacaggt agttttccag tagtgcaaat aaatttaagg
     1681 gtaagttttc cgtatgttgc atcaccttca ccctctccac tgacagaaaa tttgtgccca
     1741 ttaacatcac catctaattc aacaagaatt gggacaactc cagtgaaaag ttcttctcct
     1801 ttactcatgc tagcggcccg agcggcccga tgtttactgt agaatactaa ttgattctta
     1861 ctgaaacaag tctctatcac tgatagggac actctatata tacctggagg aagcgccgat
     1921 cctctcaatc ttccatgaca tgcccctgag cccacagaag ccggtggaac aaattggtcg
     1981 cttctttaag aatcgttaac cggaacgtta gggtacgttg aaggagcgac caagcgagcg
     2041 acgcgagcca ccgtagccaa tgtgcgagtc cagcgagctt agaaggggca agagatgggg
     2101 cccggggggt cgaggagctt ggcccattgc atacgttgta tccatatcat aatatgtaca
     2161 tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt
     2221 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     2281 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     2341 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     2401 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     2461 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     2521 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     2581 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     2641 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     2701 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     2761 aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     2821 gctgttttga cctccataga agacaccggg accgatccag cctccgcggc cccgaattca
     2881 tatgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt
     2941 cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag agcagcctac
     3001 attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca ttgagatgtt
     3061 agataggcac catactcact tttgcccttt agaaggggaa agctggcaag attttttacg
     3121 taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca
     3181 tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt
     3241 atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg tggggcattt
     3301 tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac
     3361 acctactact gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca
     3421 aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat tagaaaaaca
     3481 acttaaatgt gaaagtggat cttaaccaat tcgccctata gtgagtcgta ttacgcgcgc
     3541 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     3601 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     3661 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg atggcggcgt tagtatcgaa
     3721 tcaacagcag tatagcgacc agcattcaca tacgattgac gcatgatatt actttctgcg
     3781 cacttaactt cgcatctggg cagatgatgt cgaggcgaaa aaaaatataa atcacgctaa
     3841 catttgatta aaatagaaca actacaatat aaaaaaacta tacaaatgac aagttcttga
     3901 aaacaagaat ctttttattg tcagtactga ttaggggcag ggcatgctca tgtagagcgc
     3961 ctgctcgccg tccgaggcgg tgccgtcgta cagggcggtg tccaggccgc agagggtgaa
     4021 ccccatccgc cggtacgcgt ggatcgccgg tgcgttgacg ttggtgacct ccagccagag
     4081 gtgcccggcg ccccgctcgc gggcgaactc cgtcgcgagc cccatcaacg cgcgcccgac
     4141 cccgtgcccc cggtgctccg gggcgacctc gatgtcctcg acggtcagcc ggcggttcca
     4201 gccggagtac gagacgacca cgaagcccgc caggtcgccg tcgtccccgt acgcgacgaa
     4261 cgtccgggag tccgggtcgc cgtcctcccc gtcgtccgat tcgtcgtccg attcgtcgtc
     4321 ggggaacacc ttggtcaggg gcgggtccac cggcacctcc cgcagggtga agccgtcccc
     4381 ggtggcggtg acgcggaaga cggtgtcggt ggtgaaggac ccatccagtg cctcgatggc
     4441 ctcggcgtcc cccgggacac tggtgcggta ccggtaagcc gtgtcgtcaa gagtggtacc
     4501 catggttgtt tatgttcgga tgtgatgtga gaactgtatc ctagcaagat tttaaaagga
     4561 agtatatgaa agaagaacct cagtggcaaa tcctaacctt ttatatttct ctacaggggc
     4621 gcggcgtggg gacaattcaa cgcgtctgtg aggggagcgt ttccctgctc gcaggtctgc
     4681 agcgaggagc cgtaattttt gcttcgcgcc gtgcggccat caaaatgtat ggatgcaaat
     4741 gattatacat ggggatgtgt gggctaaata tacgggcgac agtcacatca tgcccctgag
     4801 ctgcgcacgt caagactgtc aaggagggta ttctgggcct ccatgtcgct ggccgggtga
     4861 cccggcgggg acgaggcaag ctaaacagat ctggcgcgcc ttaattaacc cggggatccg
     4921 aagggcgaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg
     4981 cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
     5041 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     5101 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5161 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5221 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5281 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5341 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5401 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5461 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5521 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5581 aacaaaaatt taacgcgaat tttaacaaaa ttcagggcgc aagggctgct aaaggaagcg
     5641 gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
     5701 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct
     5761 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc
     5821 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
     5881 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt
     5941 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     6001 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     6061 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     6121 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     6181 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     6241 gcaggatctc ctgtcatccc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     6301 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     6361 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     6421 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     6481 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     6541 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     6601 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     6661 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     6721 tcttgacgag ttcttctgaa ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6781 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6841 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6901 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6961 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     7021 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     7081 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     7141 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     7201 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     7261 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     7321 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     7381 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     7441 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     7501 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7561 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     7621 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     7681 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7741 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
